Enzymic substance and process of making the same



Patented M a, was.

I want earner errata.

Jomcm TAKAMINE, on NEW roan, N. Y." AND JOKICHI TAKAMI'NE, 3a., or current, I

- NEW JERSEY.

ENZYMIC SUBSTANCE,AND PROCESS OF MAKING THE SAME.

No Drawing.

To all whom it may concern:

Be it known that we, JoKIoHI TAKAMINE and J OKICHI TAKAMINE, Jn, subjects of the Emperor of Japan, and residing, respectively, inthe city county, and State of New York, and Clifton, county of'Passaic, State of New Jersey, have made a certain new and useful Invention in Enzymic Substance and Procem of Making the Same, of which the following is a specification.

I This invention relates to enzymic substances, and the process of making the same, and is a division in part of our application, Serial No. 213,431, filed January 23, 1918, Patent No. 1,391,219, granted September 20, 1921.

The object of our invention is to produce an enzymic substance suitable for use in various lines of industry where the enzymic property of the substance is to be utilized.

A further object of the invention is to produce an enzymic substance for use in the industrial arts, which is stable and can be preserved without deterioration.

A further object is to produce an enzymic substance of the nature referred to from a fungus growth.

Other objects of the invention will appear more fully hereinafter.

The invention consists cedure and .the product having the 0 aracteristics hereinafter more fully setforth and finally pointed out in the appended claims.

In carrying out our invention the seed spores of suitable microscopic fungi are sown on suitable culture media and propagated under suitable conditions of temperature, humidity and handling and for a suitable period of time, for the fungus to attain that stage in which it develops its greatest enzymic power. The enzymic substances contained in the fungus are then extracted by suitable solvents and by suitable treatment so as to obtain an enzymic product for use in the industrial arts, as we will more fully explain hereinafter.

'The seed spores of the fungi genera-Aspergillus, Pennecillium or Mucor; and, particularly the seed spores of the species Ewro time o'r'yzce, are suitable for the purposes of our invention.

Comminuted' or broken grains of the cereals, such as wheat, corn, oats, barley, &c.,

- and preferably such broken or comminuted of the mass is not essential and may be omit in the mode of ro- Application flled July 30, 1921. Serial No. 488,692.

grains from which the greater part of the starchy matter has been removed, leaving the broken or comminuted parts in the form of bran, shorts, middlings, &c., and particularly wheat bran, constitute suit-able media upon which the fungus may be propagated. Other media may be employed for this purpose such as slop from alcoholic distillation residue from beer brewing, oil cake, brans, slops, and residue from sugar, glucose, and starch factories, and the like.

The mas or culture medium to beemployed is first suitably moistened with water and thoroughly sterilized. This may be accomplished with steam The seed spores of the desired fungus are sown upon thesterilized mass while the latter is in a moistened state, preferably containing from thirty to sixty per cent of moisture. The sterilizing ted although a more abundant and healthier growth of fungus is secured and a; greater development of enzymic power attained by first sterilizing the culture medium. In the sterilizing operation, bacteria contained in the culture medium, and which tend to cause undesirable fermenting or decomposing aetion in the enzymic substance when produced by the subsequent treatment of the mass, are more or less destroyed or rendered inert.

After sowing the fungus seed spores upon the culture medium the entire mass is bedded up to a thickness of from six to twenty-four inches. In the case of wheat bran, shorts, middling-s, &c., the bedding up of the mass'is not essential. The mass is maintained in a moist atmosphere and at a uniform temperature not to exceed 45 C.

There are differentmethods of carrying on such' manner as to cause every portion of v the mass to be exposed to the air and to the cooling effect of the air, .thereby preventing the mass from developing too great a heat.

Inthe course of from eight to twenty hoursv the spores begin to sprout and within twenty I10 I to thirty hours the growth of fungus be- .comes abundant. Carbonic acid gas and also, to more or less extent, according to the temperature, humidity, handling, and other conditions observed during the propagation and growth of the fungus- The various enzymes are separated fro the mass. Being soluble in water this separation of the enzymes may be efiected by lixiviating the mass with water. The solution thus obtained may be concentrated in any convenient manner. For eirample', a desirable degree of concentration maybe effected by percolating the same aqueous solution through successive batches or masses of the culture medium upon which the fungus has beengrown.

Of the enzymes contained in thesolution thus obtained diastatic enzyme usually predominates, followed by proteolytic, milkcoagulating, fat-splitting, and others, cited in their relative strength.

/ The solution or extract obtained as described is suitable for use in many industrial arts, for example, for destarching woven cot ton fabrics, where utilization is made of its property of rendering starch soluble, that 1s, its diastatic property; degumming silk fibres and woven goods, where utilization is made of its proteolytic property; in bread making, where utilization is made of its powers of liquefying and saccharifying starch; and for numerous other purposes where enzymes are useful, required or desired. This solution, however, is more .or less unstable, that is, in a comparatively limited period of time decomposition sets up therein, resulting in various kinds of fermentation, which greatly impairs, if not actually destroying the valuable enzymic properties of the solution. This is a serious objection since it necessitates the use of the solution as soon as it is made and consequently a fresh quantity must be made up each time it is required for use, and this imposes a serious drawback upon the practical availability of the enzymic solution or substance.

It is among the special purposes of our invention to produce an enzymic substance of the nature referred to which is stable and can be preserved without decomposition-and fermentation and without impairing the enzymic power thereof.

We have discovered that decomposition y p portion of culture medium to effect extracand resulting fermentation inthe enzymic substance is produced-by the presence therein of undesirable spores and bacteria which are washed out from the culture medium along with the enzymes. These spores and deleterious bacteria are eliminated and removed from the solution or are rendered inert and the fermenting action thereof impaired or destroyed in accordance with our invention. This elimination of such spores, or of the efi'ects thereof may be efi'ected in various ways. One efiicacious and convenient way is to secure a concentrated solution oying the extract obtained from one tion from successive portions'of such medium until the extract contains from fifteen to thirty per cent of solid matter, where wheat bran, shorts, middlings, or combinations of such materials are employed for the culture medium. In this case the diastatic and other enzymic strengths of the extract are so increased and the acids and other products having a toxic action upon bacteria are so concentrated that livin bacteria and other organisms cannot subsist or are rendered inert and incapable of exciting their fermenting action.

In another eflicient way of attaining the desired results we proceed as follows:

To a given quantity of the enzymic solution an inert siliceous material such as infusorial earth, fullers earth, or other similar material in the proportion of one tenth of one percent to two percent is added. The

infusorial earth, or other similar material emloyed, should be first thoroughly sterilized.

he mass is thoroughly stirred or agitated and is then filtered by suitable means. The filtering operation is repeated as often as may be required until a perfectly clear transparent solution is obtained. The elimination of the spores and bacteria thus accomplished leaves a solution possessing the desired enzymic power and which is suitable for use and in which decomposition and fermentation will not occur rapidly, and which, therefore, can be preserved for a considerable period of time without deterioration.

It sometimes occurs that even after repeated filtering some deleterious bacteria still remain in the solution. To secure a still more stable solution which can be preserved without deterioration it is necessary to sterilize 0r render all or, in case of previous filtration, any remaining bacteria contained therein inert. The d'ifliculty in accomplishing this resides in the danger of impairing the enzymic power of the substance. sterilizing antiseptics such as. formaldehyde, sulphuric acid, and other suitable bactericidesmay be used for the purpose, but the danger in the use of such antiseptics is that if a sufiicient quantity of antiseptic is used to thoroughly effect. sterilization of the undesirable bacteria contained in the enzymic solution, the enzymic power of the solution is materially impaired, if not altogether destroyed. The solution may also be heated to a usual pasteurizing temperature, say to about 60 C., or higher. VVhilethis temperature is effective in killing the injurious bacteria it also acts to gereatly impair, if not destroy, the enzymic power of thesolution. In other words an antiseptic in suificient quantity, or a temperature of sufficiently high degree, to insure elimination of the injurious effects of undefired bacteria present in the solution, also greatly reduces the value of the solution by diminishing its enzymic power, if' not actually destroying such power. While, therefore, an antiseptic, such as formaldehyde, or sulphurous acid in the proportion of a small fraction of one percent, added to the clear transparent solution aids in increasing the stability ofthe solution, We have discovered, that by a novel and proper combination of antiseptic in suitable quantity, and the application of a proper amount of heat, the. joint effect of the two agencies, when properly applied under proper conditions, we are able to secure the best results in obtaining the desired effect of stability of the enzymic substances, Without injury to the enzymic power there of. The process of employing an antisep tic such as sulphuro'us acid, or the like, either alone or in combination with treatment by heat for the purpose of eliminating the deleterious effects of the bacteria from the product or rendering the same more stable, forms the subject matter in its broad scope of our said co-pending application (Patent No. 1,391,219, granted September 20, 1921) of which the present application is, in part, a division. In place of the sulphurous acid, pr that type of acid employed for this purpose, we have found that sodium fluoride or sodium benzoate, or an antiseptic of the phenol group, such as carbolic acid, cresol or a combination of the two, may be equally well, if not more efiiciently, employed; or an agent such as formaldehyde, olive oil, hylol, toluol, thymol, beta naphthol, or chloroform may be used. When darolic acid is employed chloroform may be used to advantage in combination therewith) to eliminate the odor of the carbolic acid it the same is objectionable. With this type ofsubstance obtained and treated as above described, free from contaminating or decomposing influences by properly sterilizing and sealing the containers in which it is preserved.

tains a comparatively small I in water heated to the required temperature.

After passing through a desired length ot the coil, say one hundred feet, the solution is delivered into a sterilized container,

which, when full,'is properly sealed'to exclude outer contamination.

The product obtained is a clear transparent dark amber colored liquid having a somewhat nutty taste, with a slightly musty odor, the color, taste and odor varying somewhat according to the degree of concentration of the liquid. It contains from 15% to 25% or more solid matter consisting of organic and mineral substances in about the proportion of 90% organic and 10% mineral.

t possesses strongly diastatic and other enzymic properties and has the power of converting gelatinized starch into soluble starch, and then into various forms of dextrins, and eventually into sugars. The different stages of these conversions may be rendered visible by the addition of a weak solution of iodine to the mixture of starch and the enzymic solution. The starch itself gives an insoluble blue color while the stage of soluble starch is indicated by a soluble blue color which changes into various stages of purple to brown as the converting action progresses, the color eventually disappearing when the stage of sugars is reached in the conversion. The enzymic solution when obtained from a culture medium of wheat, bran, or other similar material suitably sterilized, gives no coloration'when an alcoholic solution of gum guaiacol and hydrogen peroxide is added thereto. This is a characteristic which distinguishes this enzymic tent of the enzymic solution, whereas an enzymic solution obtained from malt conuantity of phosphate. As compared with t e ordinary malt extract of commerce the enzymic solution of our invention contains a comparatively small amount of sugar and a comparatively large amount of proteid matter.

The proteolytic properties of the enzymic substance of our invention are shown by the digesting action it exerts upon chopped meat. Within a short period of time after the enzymic substance is added to the meat,

say within about five minutes, digestionof the meat starts in, peptones being formed at first followed by amino acids, 8pc.

The milk coagulating property of the substance is easily demonstrated by the addition of a small amount of the substance, say from one to two per cent, to the milk. The

milk coagulates to whe and eventually into cheese, a good cheese orming in about one hour, the time required varying somewhat according to the temperature.

The fat-splitting property of the enzymic substance of our invention is shown by adding the same to fat which is thereby transformed into glycerin and fatty acids. This action, however, is comparatively weak.

Having now set forth the objects and nature of our invention, and the manner of carrying the same into practical use, what We claim as new and useful, and of our own invention, and desire to secure by Letters Patent, is,

1. The process which consists in propagating a fungus upon a suitable culture medium, then extracting the soluble content of the culture medium having the fungus growth thereon, then adding a phenol to the extract.

2. The process which consists in propagating a fungus upon a culture medium, then extracting the soluble content of the culture medium having the fungus growth thereon, then adding carbolic acid to the extract.

3. The process which consists in propagating a fungus upon a culture medium, then extracting the soluble content of the culture medium having the fungus growth thereon,- and then adding a phenol and chloroform to the extract.

4. The process which consists in propagatinga fungus upon a culture medium, and extracting with water the soluble content of the resulting mass, then filtering the extract, and mixing a phenol therewith in insufficient quantity to impair-the enzymic power of the solution.

5. The process which consists in propagating a fungus upon a culture medium, and

extracting with water the soluble content of the resulting mass, then filtering the extract, and mixing carbolic acid therewith in insufiicient quantity to impair the enzymic power of the solution.

6. The process which consists in propagat ing a fungus upon a. culture medium, and extracting with water the soluble content of the resulting mass, then filtering the extract, and mixing carbolic acid and chloroform therewith in insufiicient quantity to impair the enzymic power of the solution.

7. The process which consists in propagat ing 'a fungus upon a culture medium then successively extracting the soluble content of different portions of the mass with the same liquid, and finally adding a phenol to the extract.

8. The process which consists in propagating a fungus upon a culture medium, then successively extracting the soluble content of different portions of the mass with the same liquid, and finally adding carbolic acid to the extract.

. lixiviating said mass with water, and concentrating the same, phenol thereto.

11. The process which consists of propaand finally adding a gating a fungus upon a culture medium, then lixiviating said mass with water, and concentrating the same, and finally adding carbolic acid thereto.

12. The process which consists of propagating a fungus upon a culture medium, then lixiviating said mass with water, and concentrating the same, and finally adding carbolic acid and chloroform thereto.

13. As a new article of manufacture, an enzymic solution comprising an aqueous extract from a mass of culture medium on which a fungus growth hasbeen propagated and containing a phenol.

14. As a new article of manufacture, an enzymic solution comprising an aqueousextract from a mass of culture medium on which a fungus growth has been propagated and containing carbolic acid.

' 15. As a new article of manufacture, an enzymic solution comprising an aqueous extract from a mass of culture medium on which a fungus growth has been propagated and containing carbolic acid and chloroform.

16. As a new article of manufacture, a stable enzymic solution, which is clear and transparent, and free fromdecomposing or fermenting bacteria, of slightly nutty taste and musty odor, possessing strongly diastatic and other enzymic properties, gives no coloration when an alcoholic solution of gum guaiacol and hydrogen peroxide is added thereto, and contains a large amount of phosphate as a mineral constituent, a comparatively small amount of sugar and a fairly large amount of protcid organic matter, and a phenol.

17.'As a new article of manufacture, an enzymic solution comprising an aqueous extract from a mass of culture medium on which a fungus growth has been propagated, said extract being high in phosphates, possessing marked diastatic and proteolytic properties, and containing carbolic acid.

18. As a new article of manufacture, an

enzymic solution comprising an aqueous ex- 19. As a new article of manufacture, an enzymic solution comprising an aqueous extract from a mass of culture medium on which a fungus growth has been propagated, said extract beinghigh in phosphates, possessing marked diastatic and proteolytic properties, and containing carbohc acid and chloroform.

20. The process which consists in propagating a fungus upon a suitable culture medium to develop therein an enzymic substance having .diastatic and proteolytic properties, and stabilizing said enzymic sub stance with a phenol.

21. As a new article of manufacture, an 15 enzymic substance of, vegetable fungus origin possessing diastatic and proteolytic properties, and containing aheno].

In testimony whereof we ave hereunto 7 set our hands on this 26th day of July 7- A. D., 1921.

JOKICHI TAKAMINE. .JOKIC'HI TAKAMINE, JR. 

